HAL Id: hal-03217024 https://hal.archives-ouvertes.fr/hal-03217024 Submitted on 4 May 2021 HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Effcient degradation of the organic UV filter benzophenone-3 by Sphingomonas wittichii strain BP14P isolated from WWTP sludge Sonja Fagervold, Clémence Rohée, Alice Rodrigues, Didier Stien, Philippe Lebaron To cite this version: Sonja Fagervold, Clémence Rohée, Alice Rodrigues, Didier Stien, Philippe Lebaron. Effcient degradation of the organic UV filter benzophenone-3 by Sphingomonas wittichii strain BP14P iso- lated from WWTP sludge. Science of the Total Environment, Elsevier, 2021, 758, pp.143674. 10.1016/j.scitotenv.2020.143674. hal-03217024
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HAL Id: hal-03217024https://hal.archives-ouvertes.fr/hal-03217024
Submitted on 4 May 2021
HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.
Efficient degradation of the organic UV filterbenzophenone-3 by Sphingomonas wittichii strain
BP14P isolated from WWTP sludgeSonja Fagervold, Clémence Rohée, Alice Rodrigues, Didier Stien, Philippe
Lebaron
To cite this version:Sonja Fagervold, Clémence Rohée, Alice Rodrigues, Didier Stien, Philippe Lebaron. Efficientdegradation of the organic UV filter benzophenone-3 by Sphingomonas wittichii strain BP14P iso-lated from WWTP sludge. Science of the Total Environment, Elsevier, 2021, 758, pp.143674.�10.1016/j.scitotenv.2020.143674�. �hal-03217024�
acid (98 %) and molecular grade formaldehyde were obtained from Sigma-Aldrich. 100
Pure water was obtained from an Elga Purelab Flex System (Veolia LabWater STI, 101
Antony, France). Glassware was cleaned with DCM and calcinated at 450 °C for 2 102
hours to remove all traces of organic matter. 103
104
2.2 Sampling of sludge, initial culture and enrichment culture setup 105
The sludge was sampled from the Argelès-sur-mer (France) wastewater treatment 106
plant (WWTP) on August 24th, 2018, from the bottom of a clarifying tank. The 107
sludge was collected in glass mason jars and stored at 4 °C until utilized for 108
degradation experiments. The sludge contained 2765 (+/- 310) mg/L total 109
suspended solids (TSS) and the initial BP3 concentration was below the detection 110
limit for our analysis (4.6 ng/g, see Fagervold et al. (2019)). For the initial 111
degradation experiments, 100 mL sludge was mixed with 500 mL freshwater 112
mineral media (OECD301) in a 1-L glass bottle (live). BP3 was added in methanol (2 113
mg/mL) to a final concentration of 10 µg/ml. A sterile control (SC) was prepared by 114
autoclaving twice (48 hours apart) a 1-L bottle containing sludge and media at 120 115
6
°C for 20 minutes, followed by the addition of sodium azide (1 %) and then BP3. The 116
two bottles (one live and one SC) were incubated at 25 °C in the dark and were 117
systematically shaken and opened inside a fume hood twice per week to allow 118
aeration. 119
120
An enrichment culture was initiated with inoculum from the initial flask that 121
showed degradation. The final theoretical BP3 concentration was about 10 µg/mL 122
for Enrichment 1 (the first transfer), but this concentration was increased to a 123
theoretical concentration of 100 µg/mL for Enrichment 2 and subsequent 124
enrichments. To obtain a concentration of 100 µg/mL, 2 mL of a solution of BP3 in 125
acetone (2.5 mg/mL) was added to 100-mL Erlenmeyer flasks containing 2 g of inert 126
sand. The acetone was left to evaporate overnight. Mineral media (50 mL, as above) 127
was added and the flasks were autoclaved. Each month, the cultures were 128
transferred (1% vol/vol) into new flasks with sand and BP3 (see Figure 1 for 129
diagram of the experimental setup). The cultures were incubated at 25 °C in the 130
dark on a platform shaker at 100 rpm. 131
132
Fig. 1. Schematic of the experimental design. 133
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Table 1 134 Selected chemical properties and structure of the UV filters used in this study 135 (ordered by increasing molecular weight). Note the abbreviations that are used 136 throughout. 137 138
a Degradation rate at day 14, when no degradation is seen in SC, and when p < 0.05 compared to SC with a Student t-test. n.d. stands for no degradation. b Experimental data. See protocol in Fagervold et al. (2019). LOD: Limit of detection. c According to Reaxys database (https://www.reaxys.com/, accessed on June 9, 2020)
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2.3 Biodegradation assays 141
Biodegradation assays were conducted using 15-mL glass tubes with Teflon lined 142
caps (Pyrex, Analytic lab, France). The tubes were set up as for the enrichment 143
cultures, but with 0.2 g sand, 3 mL media, and care was taken so that for each batch 144
of degradation assays, the exact same amount of BP3 (around 100 µg/mL) was 145
added to the tubes. Three tubes were sacrificed for each time-point. The tubes were 146
incubated at 25 °C after inoculation. For the enrichment cultures, the inoculum for 147
the assay was 150 µL of supernatant after rigorous mixing of the enrichment 148
culture. Inoculum preparation for the biodegradation assay of strain BP14P was 149
done as follows: The strain was grown in 100 % R2B broth (Thermo Fisher 150
Scientific, Illkirch-Graffenstaden, France) for about 40 h until the culture had 151
reached an optical density at 600nm of approximately 1.0. The cell suspension was 152
centrifuged at 4200 g for 10 min and subsequently washed 2 times with minimal 153
media before a final resuspension in minimal media (4% v/v). The cultures were 154
incubated at 25 °C in the dark on a rotary shaker at 100 rpm. 155
156
2.4 BP3 extraction 157
Two different protocols were used for the extraction of BP3, one for the initial 158
biodegradation experiments and another for the biodegradation assays for 159
enrichment cultures and isolated strains. 160
161
For the initial cultures containing WWTP sludge, 5 mL slurry was taken in duplicate 162
from each flask for each timepoint, both live and SC. The slurry was centrifuged (950 163
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x g for 20 min) to separate the aqueous and solid phases. The aqueous and solid 164
phases were then extracted and analyzed separately. The supernatant (aqueous 165
phase) was transferred to a new tube and subjected to liquid/liquid extraction with 166
acidified (0.1 % formic acid) DCM. This was achieved by adding 5 mL acidified DCM 167
to the aqueous sample, mixing by vortexing and shaking overnight at 25 °C in the 168
dark. The DCM fraction was decanted off and dehydrated with Na2SO4. The DCM was 169
then evaporated using the HT-4X system (Genevac, Biopharma Technologies, 170
Diemoz, France) and the residue was dissolved in 1 mL of NMP/water (8:2, v/v) 171
before analysis. 172
173
The solid phase was extracted as described by Fagervold and coworkers (Fagervold 174
et al., 2019) with modifications. Briefly, the pellet was frozen at –80 °C, lyophilized 175
and extracted twice with 2 ml acidified (0.1% formic acid) DCM/methanol mix (1:1, 176
vol/vol) and sonicated for 10 min. The supernatants were combined, evaporated 177
and re-dissolved in NMP/water (8:2, v/v) before HPLC analysis. 178
179
For the biodegradation assays, the whole tube containing sand and liquid media was 180
sacrificed for extraction at each timepoint. DCM (3 mL) containing an exact known 181
amount of MBBT (internal standard) was added to each tube. The tubes were mixed 182
by vortexing twice for 10 seconds, 1 h apart and shaken overnight at 100 rpm at 25 183
°C in the dark. After a brief sonication, 1 mL of the DCM phase was transferred to a 184
HPLC vial and diluted 20X in DCM before HPLC analysis. 185
186
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2.5 HPLC analysis 187
The UV filters were analyzed using an Ultimate 3000TM HPLC system, equipped with 188
a DAD detector (Thermo Fisher Scientific) and a Phenomenex Kinetex Biphenyl 2.6 189
µm, 150 × 4.6 mm column as previously described (Fagervold et al., 2019) with 190
some minor modifications. The injection volume was 5 µl and the samples were 191
diluted in 100% DCM. We expanded the analysis described in Fagervold et al. 192
(2019) to include all targeted UV-filters. The calibration curves and retention times 193
of all the different UV filters (Table 1) were determined as described by Fagervold 194
and coworkers (Fagervold et al., 2019). The recovery rate was above 85 % for the 195
extraction of BP3 in the initial sludge microcosms. The recovery rate for the 196
degradation assays were variable for the different UV filters, ranging from 63 % for 197
ES to 93 % for OC. However, the extraction efficiency was repeatable for each UV 198
filter over time, which is the most important factor for the detection of degradation 199
activity. 200
201
For determination of possible BP3 degradation products, we also performed a 202
UHPLC-MS/MS analyses as described by Stien and coworkers (Stien et al., 2019). 203
The data were acquired using the Trace FinderTM 3.1 software and were processed 204
with FreeStyleTM and Compound DiscovererTM (all from Thermo Fisher Scientific). 205
206
2.6 Microbial community analysis of enrichment cultures 207
DNA was extracted using a Maxwell® 16 LEV Blood DNA Kit (Promega, 208
Charbonnières-les-Bains, France) with the Maxwell® 16 MDx Instrument (AS3000; 209
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Promega) following the manufacturer’s instructions and with an initial lysing step. 210
Briefly, 2 mL of supernatant was transferred to a microcentrifuge tube and 211
centrifuged at 14 500 x g for 10 minutes. The pellet was resuspended in 300 µL 212
MilliQ water and 300 mL of the lysing buffer from the Maxwell® 16 LEV Blood DNA 213
Kit was added to “lysing matrix B beads” (MP Biochemicals, Illkirch, France) and 214
homogenized for 20 s at 6 m/s on a FastPrep-24™ 5G Instrument (MP biochemicals). 215
After extraction, the DNA was stored at –80 °C until further analysis. 216
The composition and diversity of the bacterial communities in enrichment cultures 217
were determined by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and 218
by Illumina sequencing (RTL genomics, Texas, USA). The ARISA was performed as 219
described by Fisher et al. (Fisher MM and Triplett EW, 1999) with minor 220
modifications. Briefly, for the PCR we utilized the intergenic spacer primers 1406F 221
and 23SRY (Fisher MM and Triplett EW, 1999) and the KAPA2G Fast HotStart 222
ReadyMix (Roche, Analytic Lab, St Mathieu de Treviers, France) with the following 223
PCR cycling parameters: 94 °C for 3 minutes, 30 cycles of 94 °C for 15 s, 55 °C for 15 224
s and 72 °C for 30 s, then a final elongation of 72 °C for 1 min. The products were 225
purified, denatured and injected into a 16 capillary Applied Biosystems Sequencer 226
3130XL (Thermo Fisher Scientific) together with the internal standard 227
MapMarker® X-Rhodamine Labeled 50-1000 bp (Bioventures Inc., Tennessee, USA) 228
for the determination of peak lengths. For the analysis, individual fragment lengths 229
(within a 0.1 bp “error range”) were considered as separate Operational Taxonomic 230
Units (OTUs) and the relative abundance of each OTU was calculated by dividing the 231
area of the peak by the total area of the chromatogram after 200 bp. Furthermore, 232
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we only took into account peaks contributing to more than 1 % of the relative peak 233
area. Concerning the Illumina sequencing analysis, the DNA was sent to RTL 234
genomics where paired end, 2×300 bp MiSeq Sequencing was performed using 235
standard conditions of the sequencing provider. We opted for dual indexing and 236
used the bacterial primers 341F (CCTACGGGNGGCWGCAG) and 806R 237
(GACTACHVGGGTATCTAATCC) (Herlemann et al., 2011). The sequences were 238
processed using Qiime2 (Bolyen et al., 2019). Briefly, the sequences were quality 239
filtered, stitched, denoised and clustered using dada2. Representative sequences 240
were classified using the Naive Bayes classifier and the Silva 132 database as a 241
reference. Our two samples (Enr 11 and Enr 12) yielded about 41000 and 47000 242
sequences, of which 39000 and 44000, respectively, belonged to a single OTU. There 243
were a total of 37 OTUs in the two samples, but most were present at very low 244
relative abundances. 245
246
2.7 Strain isolation and sequencing 247
The supernatant of an actively degrading enrichment culture transferred 7 times 248
(Enrichment 7, or Enr 7) was diluted 100 fold and spread on R2B agar and 249
incubated for 3 days at 25 °C in the dark. Several colony morphologies were 250
observed and each morphotype was re-streaked on R2B agar to ensure purity. 251
These strains were tested for BP3 degradation activity and one positive strain, 252
BP14P, was further characterized. DNA was extracted as described above and 253
sequenced on the same sequencer used for ARISA. Here, we used universal bacterial 254
primers 27F and 1492R in the first PCR and then the internal primers 907R, 804F 255
13
and S8 for the sequencing reactions realized by the dideoxy reaction Sanger 256
sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo 257
Fischer) and the manufacturer's protocol. 258
259
2.8 Flow Cytometry 260
Strain BP14P cells were counted throughout the degradation assay. The glass tubes 261
were vortexed and 1 mL of cell suspension was transferred to a microcentrifuge 262
tube before adding glutaraldehyde (1% v/v final concentration). The tubes were 263
incubated at 4 °C in the dark for 15 minutes before flash freezing with liquid N2. The 264
samples were kept at –80 °C until analysis. Samples were defrosted at room 265
temperature and incubated with the nucleic acid stain SYBRGreen I (Molecular 266
Probes) for 15 min at room temperature in the dark before enumeration (Marie et 267
al., 1997) using a FACSCanto flow cytometer (BD-Biosciences) equipped with optics 268